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1.  Characterization of drug-induced transcriptional modules: towards drug repositioning and functional understanding 
Drug-induced transcriptional modules (biclusters) were identified and annotated in three human cell lines and rat liver. These were used to assess conservation across systems and to infer and experimentally validate novel drug effects and gene functions.
Biclustering of drug-induced gene expression profiles resulted in modules of drugs and genes, which were enriched in both drug and gene annotations.Identifying drug-induced transcriptional modules separately in three human cell lines and rat liver allows assessment of their conservation across model systems. About 70% of modules are conserved across cell lines, a lower bound of 15% was estimated for their conservation across organisms, and between the in vitro and in vivo systems.Drug-induced transcriptional modules can predict novel gene functions. A conserved module associated with (chole)sterol metabolism revealed novel regulators of cellular cholesterol homeostasis; 10 of them were validated in functional imaging assays.Analysis of drugs clustered into modules can give new insights into their mechanisms of action and provide leads for drug repositioning. We predicted and experimentally validated novel cell cycle inhibitors and modulators of PPARγ, estrogen and adrenergic receptors, with potential for developing new therapies against diabetes and cancer.
In pharmacology, it is crucial to understand the complex biological responses that drugs elicit in the human organism and how well they can be inferred from model organisms. We therefore identified a large set of drug-induced transcriptional modules from genome-wide microarray data of drug-treated human cell lines and rat liver, and first characterized their conservation. Over 70% of these modules were common for multiple cell lines and 15% were conserved between the human in vitro and the rat in vivo system. We then illustrate the utility of conserved and cell-type-specific drug-induced modules by predicting and experimentally validating (i) gene functions, e.g., 10 novel regulators of cellular cholesterol homeostasis and (ii) new mechanisms of action for existing drugs, thereby providing a starting point for drug repositioning, e.g., novel cell cycle inhibitors and new modulators of α-adrenergic receptor, peroxisome proliferator-activated receptor and estrogen receptor. Taken together, the identified modules reveal the conservation of transcriptional responses towards drugs across cell types and organisms, and improve our understanding of both the molecular basis of drug action and human biology.
doi:10.1038/msb.2013.20
PMCID: PMC3658274  PMID: 23632384
cell line models in drug discovery; drug-induced transcriptional modules; drug repositioning; gene function prediction; transcriptome conservation across cell types and organisms
2.  Delineation of structural domains and identification of functionally important residues in DNA repair enzyme exonuclease VII 
Nucleic Acids Research  2012;40(16):8163-8174.
Exonuclease VII (ExoVII) is a bacterial nuclease involved in DNA repair and recombination that hydrolyses single-stranded DNA. ExoVII is composed of two subunits: large XseA and small XseB. Thus far, little was known about the molecular structure of ExoVII, the interactions between XseA and XseB, the architecture of the nuclease active site or its mechanism of action. We used bioinformatics methods to predict the structure of XseA, which revealed four domains: an N-terminal OB-fold domain, a middle putatively catalytic domain, a coiled-coil domain and a short C-terminal segment. By series of deletion and site-directed mutagenesis experiments on XseA from Escherichia coli, we determined that the OB-fold domain is responsible for DNA binding, the coiled-coil domain is involved in binding multiple copies of the XseB subunit and residues D155, R205, H238 and D241 of the middle domain are important for the catalytic activity but not for DNA binding. Altogether, we propose a model of sequence–structure–function relationships in ExoVII.
doi:10.1093/nar/gks547
PMCID: PMC3439923  PMID: 22718974
4.  2′-O-ribose methylation of cap2 in human: function and evolution in a horizontally mobile family 
Nucleic Acids Research  2011;39(11):4756-4768.
The 5′ cap of human messenger RNA consists of an inverted 7-methylguanosine linked to the first transcribed nucleotide by a unique 5′–5′ triphosphate bond followed by 2′-O-ribose methylation of the first and often the second transcribed nucleotides, likely serving to modify efficiency of transcript processing, translation and stability. We report the validation of a human enzyme that methylates the ribose of the second transcribed nucleotide encoded by FTSJD1, henceforth renamed HMTR2 to reflect function. Purified recombinant hMTr2 protein transfers a methyl group from S-adenosylmethionine to the 2′-O-ribose of the second nucleotide of messenger RNA and small nuclear RNA. Neither N7 methylation of the guanosine cap nor 2′-O-ribose methylation of the first transcribed nucleotide are required for hMTr2, but the presence of cap1 methylation increases hMTr2 activity. The hMTr2 protein is distributed throughout the nucleus and cytosol, in contrast to the nuclear hMTr1. The details of how and why specific transcripts undergo modification with these ribose methylations remains to be elucidated. The 2′-O-ribose RNA cap methyltransferases are present in varying combinations in most eukaryotic and many viral genomes. With the capping enzymes in hand their biological purpose can be ascertained.
doi:10.1093/nar/gkr038
PMCID: PMC3113572  PMID: 21310715
5.  Structural basis for the methylation of G1405 in 16S rRNA by aminoglycoside resistance methyltransferase Sgm from an antibiotic producer: a diversity of active sites in m7G methyltransferases 
Nucleic Acids Research  2010;38(12):4120-4132.
Sgm (Sisomicin-gentamicin methyltransferase) from antibiotic-producing bacterium Micromonospora zionensis is an enzyme that confers resistance to aminoglycosides like gentamicin and sisomicin by specifically methylating G1405 in bacterial 16S rRNA. Sgm belongs to the aminoglycoside resistance methyltransferase (Arm) family of enzymes that have been recently found to spread by horizontal gene transfer among disease-causing bacteria. Structural characterization of Arm enzymes is the key to understand their mechanism of action and to develop inhibitors that would block their activity. Here we report the structure of Sgm in complex with cofactors S-adenosylmethionine (AdoMet) and S-adenosylhomocysteine (AdoHcy) at 2.0 and 2.1 Å resolution, respectively, and results of mutagenesis and rRNA footprinting, and protein-substrate docking. We propose the mechanism of methylation of G1405 by Sgm and compare it with other m7G methyltransferases, revealing a surprising diversity of active sites and binding modes for the same basic reaction of RNA modification. This analysis can serve as a stepping stone towards developing drugs that would specifically block the activity of Arm methyltransferases and thereby re-sensitize pathogenic bacteria to aminoglycoside antibiotics.
doi:10.1093/nar/gkq122
PMCID: PMC2896518  PMID: 20194115
6.  A putative mobile genetic element carrying a novel type IIF restriction-modification system (PluTI) 
Nucleic Acids Research  2010;38(9):3019-3030.
Genome comparison and genome context analysis were used to find a putative mobile element in the genome of Photorhabdus luminescens, an entomopathogenic bacterium. The element is composed of 16-bp direct repeats in the terminal regions, which are identical to a part of insertion sequences (ISs), a DNA methyltransferase gene homolog, two genes of unknown functions and an open reading frame (ORF) (plu0599) encoding a protein with no detectable sequence similarity to any known protein. The ORF (plu0599) product showed DNA endonuclease activity, when expressed in a cell-free expression system. Subsequently, the protein, named R.PluTI, was expressed in vivo, purified and found to be a novel type IIF restriction enzyme that recognizes 5′-GGCGC/C-3′ (/ indicates position of cleavage). R.PluTI cleaves a two-site supercoiled substrate at both the sites faster than a one-site supercoiled substrate. The modification enzyme homolog encoded by plu0600, named M.PluTI, was expressed in Escherichia coli and shown to protect DNA from R.PluTI cleavage in vitro, and to suppress the lethal effects of R.PluTI expression in vivo. These results suggested that they constitute a restriction–modification system, present on the putative mobile element. Our approach thus allowed detection of a previously uncharacterized family of DNA-interacting proteins.
doi:10.1093/nar/gkp1221
PMCID: PMC2875022  PMID: 20071747
7.  Insights into the structure, function and evolution of the radical-SAM 23S rRNA methyltransferase Cfr that confers antibiotic resistance in bacteria 
Nucleic Acids Research  2009;38(5):1652-1663.
The Cfr methyltransferase confers combined resistance to five classes of antibiotics that bind to the peptidyl tranferase center of bacterial ribosomes by catalyzing methylation of the C-8 position of 23S rRNA nucleotide A2503. The same nucleotide is targeted by the housekeeping methyltransferase RlmN that methylates the C-2 position. Database searches with the Cfr sequence have revealed a large group of closely related sequences from all domains of life that contain the conserved CX3CX2C motif characteristic of radical S-adenosyl-l-methionine (SAM) enzymes. Phylogenetic analysis of the Cfr/RlmN family suggests that the RlmN subfamily is likely the ancestral form, whereas the Cfr subfamily arose via duplication and horizontal gene transfer. A structural model of Cfr has been calculated and used as a guide for alanine mutagenesis studies that corroborate the model-based predictions of a 4Fe–4S cluster, a SAM molecule coordinated to the iron–sulfur cluster (SAM1) and a SAM molecule that is the putative methyl group donor (SAM2). All mutations at predicted functional sites affect Cfr activity significantly as assayed by antibiotic susceptibility testing and primer extension analysis. The investigation has identified essential amino acids and Cfr variants with altered reaction mechanisms and represents a first step towards understanding the structural basis of Cfr activity.
doi:10.1093/nar/gkp1142
PMCID: PMC2836569  PMID: 20007606
8.  Cell death upon epigenetic genome methylation: a novel function of methyl-specific deoxyribonucleases 
Genome Biology  2008;9(11):R163.
The McrBC methyl-specific deoxyribonuclease from Escherichia coli can respond to genome methylation by host killing.
Background
Alteration in epigenetic methylation can affect gene expression and other processes. In Prokaryota, DNA methyltransferase genes frequently move between genomes and present a potential threat. A methyl-specific deoxyribonuclease, McrBC, of Escherichia coli cuts invading methylated DNAs. Here we examined whether McrBC competes with genome methylation systems through host killing by chromosome cleavage.
Results
McrBC inhibited the establishment of a plasmid carrying a PvuII methyltransferase gene but lacking its recognition sites, likely through the lethal cleavage of chromosomes that became methylated. Indeed, its phage-mediated transfer caused McrBC-dependent chromosome cleavage. Its induction led to cell death accompanied by chromosome methylation, cleavage and degradation. RecA/RecBCD functions affect chromosome processing and, together with the SOS response, reduce lethality. Our evolutionary/genomic analyses of McrBC homologs revealed: a wide distribution in Prokaryota; frequent distant horizontal transfer and linkage with mobility-related genes; and diversification in the DNA binding domain. In these features, McrBCs resemble type II restriction-modification systems, which behave as selfish mobile elements, maintaining their frequency by host killing. McrBCs are frequently found linked with a methyltransferase homolog, which suggests a functional association.
Conclusions
Our experiments indicate McrBC can respond to genome methylation systems by host killing. Combined with our evolutionary/genomic analyses, they support our hypothesis that McrBCs have evolved as mobile elements competing with specific genome methylation systems through host killing. To our knowledge, this represents the first report of a defense system against epigenetic systems through cell death.
doi:10.1186/gb-2008-9-11-r163
PMCID: PMC2614495  PMID: 19025584
9.  Type II restriction endonuclease R.Hpy188I belongs to the GIY-YIG nuclease superfamily, but exhibits an unusual active site 
Background
Catalytic domains of Type II restriction endonucleases (REases) belong to a few unrelated three-dimensional folds. While the PD-(D/E)XK fold is most common among these enzymes, crystal structures have been also determined for single representatives of two other folds: PLD (R.BfiI) and half-pipe (R.PabI). Bioinformatics analyses supported by mutagenesis experiments suggested that some REases belong to the HNH fold (e.g. R.KpnI), and that a small group represented by R.Eco29kI belongs to the GIY-YIG fold. However, for a large fraction of REases with known sequences, the three-dimensional fold and the architecture of the active site remain unknown, mostly due to extreme sequence divergence that hampers detection of homology to enzymes with known folds.
Results
R.Hpy188I is a Type II REase with unknown structure. PSI-BLAST searches of the non-redundant protein sequence database reveal only 1 homolog (R.HpyF17I, with nearly identical amino acid sequence and the same DNA sequence specificity). Standard application of state-of-the-art protein fold-recognition methods failed to predict the relationship of R.Hpy188I to proteins with known structure or to other protein families. In order to increase the amount of evolutionary information in the multiple sequence alignment, we have expanded our sequence database searches to include sequences from metagenomics projects. This search resulted in identification of 23 further members of R.Hpy188I family, both from metagenomics and the non-redundant database. Moreover, fold-recognition analysis of the extended R.Hpy188I family revealed its relationship to the GIY-YIG domain and allowed for computational modeling of the R.Hpy188I structure. Analysis of the R.Hpy188I model in the light of sequence conservation among its homologs revealed an unusual variant of the active site, in which the typical Tyr residue of the YIG half-motif had been substituted by a Lys residue. Moreover, some of its homologs have the otherwise invariant Arg residue in a non-homologous position in sequence that nonetheless allows for spatial conservation of the guanidino group potentially involved in phosphate binding.
Conclusion
The present study eliminates a significant "white spot" on the structural map of REases. It also provides important insight into sequence-structure-function relationships in the GIY-YIG nuclease superfamily. Our results reveal that in the case of proteins with no or few detectable homologs in the standard "non-redundant" database, it is useful to expand this database by adding the metagenomic sequences, which may provide evolutionary linkage to detect more remote homologs.
doi:10.1186/1472-6807-8-48
PMCID: PMC2630997  PMID: 19014591
10.  MODOMICS: a database of RNA modification pathways. 2008 update 
Nucleic Acids Research  2008;37(Database issue):D118-D121.
MODOMICS, a database devoted to the systems biology of RNA modification, has been subjected to substantial improvements. It provides comprehensive information on the chemical structure of modified nucleosides, pathways of their biosynthesis, sequences of RNAs containing these modifications and RNA-modifying enzymes. MODOMICS also provides cross-references to other databases and to literature. In addition to the previously available manually curated tRNA sequences from a few model organisms, we have now included additional tRNAs and rRNAs, and all RNAs with 3D structures in the Nucleic Acid Database, in which modified nucleosides are present. In total, 3460 modified bases in RNA sequences of different organisms have been annotated. New RNA-modifying enzymes have been also added. The current collection of enzymes includes mainly proteins for the model organisms Escherichia coli and Saccharomyces cerevisiae, and is currently being expanded to include proteins from other organisms, in particular Archaea and Homo sapiens. For enzymes with known structures, links are provided to the corresponding Protein Data Bank entries, while for many others homology models have been created. Many new options for database searching and querying have been included. MODOMICS can be accessed at http://genesilico.pl/modomics.
doi:10.1093/nar/gkn710
PMCID: PMC2686465  PMID: 18854352
11.  The YqfN protein of Bacillus subtilis is the tRNA: m1A22 methyltransferase (TrmK) 
Nucleic Acids Research  2008;36(10):3252-3262.
N1-methylation of adenosine to m1A occurs in several different positions in tRNAs from various organisms. A methyl group at position N1 prevents Watson–Crick-type base pairing by adenosine and is therefore important for regulation of structure and stability of tRNA molecules. Thus far, only one family of genes encoding enzymes responsible for m1A methylation at position 58 has been identified, while other m1A methyltransferases (MTases) remain elusive. Here, we show that Bacillus subtilis open reading frame yqfN is necessary and sufficient for N1-adenosine methylation at position 22 of bacterial tRNA. Thus, we propose to rename YqfN as TrmK, according to the traditional nomenclature for bacterial tRNA MTases, or TrMet(m1A22) according to the nomenclature from the MODOMICS database of RNA modification enzymes. tRNAs purified from a ΔtrmK strain are a good substrate in vitro for the recombinant TrmK protein, which is sufficient for m1A methylation at position 22 as are tRNAs from Escherichia coli, which natively lacks m1A22. TrmK is conserved in Gram-positive bacteria and present in some Gram-negative bacteria, but its orthologs are apparently absent from archaea and eukaryota. Protein structure prediction indicates that the active site of TrmK does not resemble the active site of the m1A58 MTase TrmI, suggesting that these two enzymatic activities evolved independently.
doi:10.1093/nar/gkn169
PMCID: PMC2425500  PMID: 18420655
12.  Structural analysis of human 2′-O-ribose methyltransferases involved in mRNA cap structure formation 
Nature Communications  2014;5:3004.
The 5′ cap of human messenger RNA contains 2′-O-methylation of the first and often second transcribed nucleotide that is important for its processing, translation and stability. Human enzymes that methylate these nucleotides, termed CMTr1 and CMTr2, respectively, have recently been identified. However, the structures of these enzymes and their mechanisms of action remain unknown. In the present study, we solve the crystal structures of the active CMTr1 catalytic domain in complex with a methyl group donor and a capped oligoribonucleotide, thereby revealing the mechanism of specific recognition of capped RNA. This mechanism differs significantly from viral enzymes, thus providing a framework for their specific targeting. Based on the crystal structure of CMTr1, a comparative model of the CMTr2 catalytic domain is generated. This model, together with mutational analysis, leads to the identification of residues involved in RNA and methyl group donor binding.
Human mRNA transcripts possess a 5' cap structure that is modified by methylation. Here, Smietanski et al. present the structures of human methyltransferases responsible for this reaction, revealing key differences to their viral counterparts and thereby providing a framework for targeted drug design.
doi:10.1038/ncomms4004
PMCID: PMC3941023  PMID: 24402442

Results 1-12 (12)